Glucose is transported across the plasma membrane of mammalian cells by a family of membrane glycoproteins comprised of five members. The five glucose transporter isoforms are expressed in a tissue-specific manner and exhibit distinct kinetic and regulatory properties. In the preceding project period we discovered and cloned a novel glucose transporter isoform, designated GLUT4, that is the major transporter expressed in the insulin-sensitive tissues, fat and muscle. Glucose transport in these tissues is rapidly stimulated by elevated blood insulin levels, a process that is critical to the maintenance of normal glucose homeostasis. Current evidence suggests that a disruption in insulin-stimulated glucose transport in muscle is the basis for the insulin-resistance associated with non- insulin-dependent diabetes mellitus (NIDDM). It has been proposed that a genetic defect in the regulation of GLUT4 is a major predisposing factor to NIDDM. The long-term goal of this proposal is to elucidate the molecular mechanism of insulin-stimulated glucose transport and to define the physiological roles of the GLUT1 and GLUT4 glucose transporter isoforms in the maintenance of mammalian glucose homeostasis. To accomplish this goal, the following three specific aims will be undertaken: l. To determine the structural feature of GLUT4 that targets it to an insulin-responsive intracellular membrane compartment. Chimeric glucose transporter cDNAs encoding GLUT1/GLUT4 fusion proteins will be constructed and expressed in 3T3LI cells. The GLUT4 targeting sequence will be identified by determining the subcellular distribution of the chimeric glucose transporter molecules by confocal immunofluorescence microscopy. 2. To determine the mechanisms underlying the long-term regulation of glucose transport in 3T3Ll adipocytes. Nuclear run-on assays will be used to investigate the possible transcriptional effects of glucose deprivation, insulin, and growth hormone on transporter gene expression. Cis-acting elements and trans-acting factors involved in the transcriptional regulation will be identified and characterized. The role of transporter glycosylation in the glucose deprivation response will be explored by creation of a mutant cDNA encoding a GLUT1 transporter lacking the glycosylated asparagine residue and examining the subcellular targeting and intrinsic activity of the mutant in transfected 3T3Ll adipocytes. 3. To determine the physiologic roles of GLUT1 and GLUT4 in whole-body glucose disposal by analysis of transgenic mice overexpressing these proteins. GLUT1 and GLUT4 will be overexpressed in skeletal muscle of transgenic mice by using cDNA constructions in which transcription kinase promoter and enhancer sequences. The effect of the GLUT1 and blood glucose levels and insulin-sensitivity will be examined in streptozotocin-diabetic and genetically diabetic (db/db) mice.